1) Mutations in human lung adenocarcinomas Will Lockwood (with the earlier help of Kreshnik Zejnullahu) has been directing the sequencing of unusual lung adenocarcinomas, with emphasis on tumors arising in patients who have never smoked, in a partnership with the NHGRI Sequencing Core and the University of British Columbia, to search for novel mutations that drive tumor development. Whole-exome sequences have been integrated with pre-existing data about single nucleotide polymorphisms, copy number variation, CGH, DNA methylation, and mRNA expression. Even in a relatively small cohort of tumors, we have identified a few novel candidate driver genes, in addition to well-established ones, and these are currently being assessed in a larger cohort of 100 lung tumor/normal pairs. Functional validation of candidates, such as alternatively spliced MET genes and novel cancer genes, is being undertaken in cell lines and in mouse models of lung adenocarcinoma. This work is being continued through a collaboration with Will Lockwood, who is now on the faculty at UBC. 2) Role of RNA processing in lung adenocarcinogenesis Characterization of cancer genomes by TCGA and other teams over the past few years has revealed that cancer cells often harbor mutations in splicing signals and in genes encoding known splicing factors. These mutations are likely to contribute to neoplasia by generating abnormal RNAs and proteins, although the critical features of this putative oncogenic mechanism have not yet been defined. Dennis Fei is attempting to characterize the oncogenic properties of mutant proteins that regulate RNA splicing in some human cancers. He is focusing on a mutation (S34F) in the splicing factor U2AF1 that has been repeatedly observed in lung adenocarcinoma, myeloid dysplastic syndrome, and acute myeloid leukemia. In efforts to seek oncogenic effects of the mutant gene, he has introduced it into cultured human lung cell and myeloid lines, and he has made mice in which the mutant gene can be activated by the Cre recombinase. In these biological settings, with some help from Jovian Yu, he is now looking for changes in spliced RNAs, for changes in the development and behavior of lung and hematopoietic cells, and for effects on oncogenesis promoted by other cancer genes, such as mutant EGFR or KRAS (for lung adenocarcinoma) and drivers of myeloid dysplasias and cancers. Recent results suggest that a large subset of mRNAs are aberrantly spliced and that expression of U2AF1S34F in mice leads to leukopenia and arrested differentiation. Some of these findings are being further pursued in collaboration with Paul Liu (NHGRI) and Tim Graubert (Washington University). 3)Mutual exclusivity of KRAS and EGFR mutations in human lung adenocarcinomas Although lung adenocarcinomas frequently contain KRAS or EGFR mutations (in about 25 and 10% of tumors, respectively), tumors containing mutations in both genes have not be observed. Statistical evaluation argues that such mutual exclusivity has not occurred by chance. Not only are the two genes thought to be acting in the same signaling pathways, which may explain a lack of additive or synergistic effects, but co-existence of mutations in both genes is likely to be detrimental and thus selected against. Will Lockwood and Arun Unni have carried out a set of experiments in mice with inducible mutant EGFR and KRAS transgenes and in cell lines in which the two mutant genes can be expressed or introduced by viral vectors to attempt to document the predicted incompatibility and to understand and perhaps exploit the predicted detrimental effects. These experiments show that the two oncogenes are synthetically lethal, causing macropinocytosis through probable activation of the MAP kinase pathway. Other examples of this form of lethality and applications in cancer therapy are being pursued. 4)Inflammation, immune response, and lung tumorigenesis The immune system can promote or retard cancer progression in different settings. Arun Unni has chosen to better understand this interplay by using our mutant EGFR-driven mouse models of lung adenocarcinoma. He has observed early recruitment of macrophages to the lung parenchyma after induction of mutant EGFR. Experiments in various cell lines suggest that signaling by mutant EGFR is capable of driving an inflammatory response by induction of expression of cytokines. By isolating clonal lines expressing different levels of EGFR, he has been able to identify a threshold for induction of a set of factors, including cytokines that mediate immune responsiveness and a readily measured cell surface protein, PD-L1, a ligand that reduces the activity of tumor-reactive T cells. These signals are relayed from activated EGFR via STAT1, simulating an interferon response. Their significance in the progression and therapy of cancers, including immunotherapy is under study. 5)Using insertional mutagens to define molecular events that cooperate with known lung cancer driver gene. Pang Fan (a former post-doc, still at MSKCC), Will Lockwood, and Dennis Fei have adapted the Sleeping Beauty (SB) transposition system to generate mice that express both the SB transposase and transposon, along with mutant EGFR alleles, specifically in type 2 airway epithelial cells after doxycycline treatment. Although SB transposition does not appear to accelerate tumorigenesis by mutant EGFR, we have observed some tumors in mice lacking EGFR transgenes, and we are currently examining the SB insertion sites in tumors arising in mice with and without mutant EGFR, seeking genes that initiate or potentiate oncogenic growth. In addition, Dennis Fei has recently started using the SB system to study another important type of lung cancer arising in small (neuro-endocrine) cells. Collaborative projects Our group is involved in a variety of collaborations, most initiated before we arrived at the NIH and conducted largely in other laboratories. Most involve studies of lung carcinogenesis in our mouse models and have produced some of the publications in the list below. Further details are available upon request.